ARTHRITIS & RHEUMATISM Vol. 46, No. 6, June 2002, pp 1683–1688 DOI 10.1002/art.10327 © 2002, American College of Rheumatology Fenofibrate Inhibits Reactive Amyloidosis in Mice Takehiro Murai,1 Toshiyuki Yamada,2 Takashi Miida,1 Katsumitsu Arai,1 Naoto Endo,1 and Tadamasa Hanyu1 (4,5). SAA, an acute-phase reactant, is synthesized predominantly in the liver after stimulation by inflammation-related cytokines, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor ␣ (TNF␣) (6). Although SAA is insoluble by itself, it circulates in the blood as an apolipoprotein of high-density lipoprotein (HDL). When SAA is dissociated from HDL and partially degraded, it is deposited as AA amyloid fibrils in the extracellular space of vital tissues (5,7), leading to renal failure and gastrointestinal tract dysfunction (8). Prolonged hyperproduction of SAA with chronic inflammation is the primary cause of reactive amyloidosis, although other factors, such as genetic background (8,9) and impaired processing of SAA (5,10), may contribute to amyloidogenesis. Fibrate derivatives, such as bezafibrate and fenofibrate, have been used to treat hypertriglyceridemia or combined hyperlipidemia. Their main effect is to reduce levels of triglycerides rather than cholesterol (11,12). Recent studies have revealed that fibrates regulate not only lipid metabolism, but also inflammation (13). Fibrates were shown to decrease plasma concentrations of inflammatory cytokines, such as IL-6, TNF␣, and interferon-␥, as well as concentrations of fibrinogen and C-reactive protein, in patients with atherosclerosis (14,15). In this study, we examined whether fenofibrate would inhibit AA amyloidosis by reducing SAA levels in a mouse model. We quantitated the amyloid deposits and SAA concentrations in mice with and without treatment. Objective. To examine the effects of the lipidlowering agent fenofibrate on experimental AA amyloidosis and on serum amyloid A (SAA) levels. Methods. Fenofibrate was administered orally in a mouse model of amyloidosis, which is induced by injections of amyloid-enhancing factor and Freund’s complete adjuvant. Fenofibrate was given for 3 weeks, including a 1-week course before induction of amyloidosis. Splenic amyloid deposits were evaluated histologically, and SAA levels were measured. Results. Fenofibrate inhibited the formation of splenic amyloid deposits and suppressed the elevation of SAA levels. Conclusion. Fenofibrate inhibits experimental amyloidosis by reducing levels of the precursor SAA. Reactive AA amyloidosis is a complication of chronic inflammatory disease which compromises quality of life and worsens the long-term prognosis. In an autopsy study in Japan, rheumatoid arthritis (RA) was present in 61.4% of subjects with reactive amyloidosis, and 25.2% of subjects with RA had this complication. Moreover, reactive amyloidosis was the cause of death in 12.5% of RA patients (1). In studies of gastrointestinal tract and renal biopsy samples, amyloid deposits were present in 13.3% and 19%, respectively, of RA patients (2,3). Reactive amyloidosis is caused by deposits of insoluble fibrils consisting of AA amyloid protein, which is derived from its precursor, serum amyloid A (SAA) Supported by grant-in-aid 13671496 for scientific research from the Japan Society for the Promotion of Science. 1 Takehiro Murai, MD, Takashi Miida, MD, Katsumitsu Arai, MD, Naoto Endo, MD, Tadamasa Hanyu, MD: Niigata University School of Medicine, Niigata, Japan; 2Toshiyuki Yamada, MD: Juntendo University School of Medicine, Tokyo, Japan. Address correspondence and reprint requests to Takashi Miida, MD, Department of Laboratory Medicine, Niigata University School of Medicine, Asahimachi 1-757, Niigata 951-8510, Japan. E-mail: [email protected] Submitted for publication June 12, 2001; accepted in revised form February 21, 2002. MATERIALS AND METHODS Agents. Fenofibrate and fenofibric acid (FA) in bulk were kindly provided by Fournier Laboratories (Dijon, France). FA is the active metabolite of fenofibrate and its major form in the circulation. Animals. A total of 60 female CBA/Jn mice (age 8 weeks, weight 20–24 gm) were purchased from Charles River Japan (Kanagawa, Japan) and maintained under specific pathogen–free conditions in our animal facility. We housed 5 1683 1684 mice per cage and fed them with regular chow and water ad libitum for 1 week to stabilize their metabolic condition. Thus, 9-week-old mice were used in our experiments. Preparation of fenofibrate-containing diet. Regular mice chow pellets were ground into powder using a food processor and uniformly mixed with fenofibrate to create 0.05%, 0.1%, and 0.2% mixtures (weight/weight). We added sterile distilled water to the individual mixture to form small pellets, which were allowed to dry completely at room temperature. Since each mouse consumed 3.0–3.5 gm of chow/day, dosages of 0.05%, 0.1%, and 0.2% fenofibrate corresponded to 75, 150, and 300 mg of fenofibrate/kg/day. These concentrations of fenofibrate were determined by previous experiments using rodents (16,17). Preparation for amyloid induction. Amyloidenhancing factor (AEF) and Freund’s complete adjuvant (CFA) were used for induction of experimental AA amyloidosis. AEF can dramatically shorten the lag phase of amyloid deposition in mice. Its chemical nature has not been clearly identified, but microfibrils or fibril-forming peptides in the preparation are believed to serve as a nidus for further amyloid formation (18). AEF was prepared as glycerol extracts of spleen from amyloidotic animals as previously described (19). CFA was purchased from Difco (Detroit, MI). Protocol. Mice were divided into 2 groups, AEF/ CFA⫺ and AEF/CFA⫹, depending on whether amyloidosis was induced. These 2 groups were subdivided, based on the dosage of fenofibrate, into 2 (0% and 0.2% fenofibrate) and 4 (0%, 0.05%, 0.1%, and 0.2% fenofibrate) subgroups, respectively. Each subgroup contained 10 mice. Amyloidosis was induced as follows: on day 0, AEF (0.2 ml) was injected intraperitoneally. On day 1, a single intraperitoneal injection of CFA (0.5 ml) was given. No further injections were needed. Mice were fed the indicated fenofibrate-containing diet or regular chow for 3 weeks (from day ⫺7 to day 14). To confirm that the doses of fenofibrate were consistent, the food intake of mice in each cage was recorded daily. All mice were bled from the retroorbital venous plexus under anesthesia on days ⫺7, 0, 2, and 7. On day 14 (the end of the experiments), all mice were killed, and the final blood samples were obtained by cardiac puncture. Serum was isolated by centrifugation and stored at ⫺80°C until assayed. Livers and spleens were also collected for histologic examination. Measurement of FA concentration. FA concentrations in plasma obtained on day 14 were measured by highperformance liquid chromatography (HPLC). First, either the internal standard (flurbiprofen; Kaken Pharmaceutical, Tokyo, Japan) or the FA standard was added to each plasma sample. Second, the sample containing the internal standard was treated with 0.5M HCl/n-hexane/ethyl acetate (1/9/1 volume/ volume/volume). The organic phase was mixed with 0.1M Na2HPO4. Then, the liquid phase was treated with 0.5M HCl/n-hexane/ethyl acetate (1/19/1 v/v/v). The organic phase was dried down and dissolved in methanol solution. Finally, the extract was separated by HPLC (LC-10A system and a C-R7A plus data processor; Shimadzu, Kyoto, Japan) using an analytical column (CapCell Pak C18 SG120, 150 ⫻ 4.6–mm inner diameter; Shiseido, Tokyo, Japan) at 35°C with ultraviolet detection (254 nm). The mobile phase consisted of a mixture of 0.02M sodium citrate buffer (pH 3.7)/acetonitrile (6/4 v/v). The MURAI ET AL Figure 1. Plasma fenofibric acid (FA) concentration on day 14. The FA level correlated positively with the dose of fenofibrate in the study diet. Values are the mean and SEM. AEF ⫽ amyloid-enhancing factor; CFA ⫽ Freund’s complete adjuvant; ND ⫽ not detectable. plasma FA concentration was calculated from the peak area ratio of FA to the internal standard. Evaluation of amyloid deposits. After fixing the liver and spleen in 10% buffered formaldehyde, we made paraffinembedded sections and stained them with hematoxylin and eosin (H&E) and Congo red. Each splenic section was evaluated histologically for the severity of amyloid deposition, as follows: 0 ⫽ no detectable amyloid, 1 ⫽ mild amyloid deposits mainly in the perifollicular zone, 2 ⫽ moderate deposits, 3 ⫽ abundant deposits but an intact follicular structure, and 4 ⫽ severe deposits with destruction of the follicular structure. Measurement of SAA, IL-6, and triglycerides in plasma. SAA concentrations were measured in plasma samples on days –7, 0, 2, 7, and 14 by an enzyme-linked immunosorbent assay (ELISA) as previously described (5). IL-6 and triglyceride concentrations were measured in plasma samples on day 14 by ELISA (Endogen, Woburn, MA) and by an enzymatic method (L-type Wako TG-H; Wako, Osaka, Japan), respectively. Statistical analysis. All data are expressed as the mean and SEM. The Mann-Whitney rank sum test was used to determine the level of significance between group means. P values less than 0.05 were considered significant. RESULTS FA concentration. On day 14, the plasma FA level correlated positively with the dose of fenofibrate in the study diet (Figure 1). In addition, the FA levels in mice receiving 0.2% fenofibrate were similar in the AEF/CFA⫺ and AEF/CFA⫹ groups. General findings. Fenofibrate markedly inhibited the macroscopic inflammatory response induced by AEF/CFA injection. Although ascites and intraperitoneal adhesions were always seen in the AEF/CFA⫹ EFFECT OF FENOFIBRATE ON AMYLOIDOSIS 1685 Figure 2. Hematoxylin and eosin–stained sections of spleen from mice with induced amyloidosis that were fed regular chow containing either (0% fenofibrate (A), 0.05% fenofibrate (B), or 0.2% fenofibrate (C). The amyloid deposits in these sections correspond to severity grades 4, 2, and 0, respectively (see Materials and Methods). (Original magnification ⫻ 100.) groups, fenofibrate clearly suppressed the severity of the inflammatory response. On H&E-stained sections, peroxisome formation was not observed in the liver in any mice. Amyloid examination. Histologic examination showed that fenofibrate markedly prevented the formation of amyloid deposits in the spleen. In the AEF/ CFA⫺ group, there were no amyloid deposits in any mice (data not shown). In the AEF/CFA⫹ group, we found abundant amyloid deposits and severe destruction of the follicular structure in mice receiving 0% fenofibrate (Figure 2A). In contrast, there were far fewer amyloid deposits in fenofibrate-treated mice (Figures 2B and C). The average severity score for amyloid deposition was highest in mice that received 0% fenofibrate and lowest in mice that received 0.2% fenofibrate (Table 1). This inhibitory effect on amyloid deposition was apparently dependent on the fenofibrate dose. Changes in SAA concentration. Fenofibrate also inhibited the elevation of SAA levels in mice after AEF/CFA injections. In the AEF/CFA⫺ group, there was no change in the SAA concentration during the entire experiment in any mouse (data not shown). In the AEF/CFA⫹ group, SAA concentrations gradually increased after injection (Figure 3). However, this AEF/ CFA-induced increase in SAA concentration was suppressed in a dose-dependent manner in mice that received 0.05%, 0.1%, and 0.2% fenofibrate compared with those that received 0% fenofibrate. Plasma IL-6 and triglyceride concentrations. The IL-6 level on day 14 was markedly higher in the AEF/ CFA⫹ group than in the AEF/CFA⫺ group Table 1. mice* Histologic evaluation of amyloid deposits in the spleen of Group, fenofibrate dose (n) AEF/CFA⫺ 0% (10) 0.2% (8) AEF/CFA⫹ 0% (10) 0.05% (9) 0.1% (10) 0.2% (10) Severity score† 0 1 2 3 4 10 8 2 5 Mean ⫾ SEM severity score 0 0 1 4 5 5 2 8 3 2 2 3.2 ⫾ 0.1 2.2 ⫾ 0.2‡ 1.4 ⫾ 0.3‡ 0.5 ⫾ 0.2§ * AEF ⫽ amyloid-enhancing factor; CFA ⫽ Freund’s complete adjuvant. † Values are the number of mice with the severity score (0 ⫽ no detectable amyloid, 1 ⫽ mild amyloid deposits mainly in the perifollicular zone, 2 ⫽ moderate deposits, 3 ⫽ abundant deposits but an intact follicular structure, 4 ⫽ severe deposits with destruction of the follicular structure). ‡ P ⬍ 0.005 versus mice that received 0% fenofibrate in the same group. § P ⬍ 0.0001 versus mice that received 0% fenofibrate in the same group. 1686 MURAI ET AL Table 3. Effect of fenofibrate on plasma triglyceride concentrations* Group, fenofibrate dose (n) AEF/CFA⫺ 0% (10) 0.2% (8) AEF/CFA⫹ 0% (10) 0.05% (9) 0.1% (10) 0.2% (10) Triglycerides, mg/dl 114 ⫾ 27 13 ⫾ 2† 46 ⫾ 5 20 ⫾ 3‡ 14 ⫾ 2† 16 ⫾ 1† * Values are the mean ⫾ SEM (see Table 1 for definitions). † P ⬍ 0.0005 versus mice that received 0% fenofibrate in the same group. ‡ P ⬍ 0.001 versus mice that received 0% fenofibrate in the same group. Figure 3. Effect of treatment with fenofibrate on serum amyloid A (SAA) concentrations before and after induction of amyloidosis. All mice were fed regular chow or chow containing fenofibrate for 3 weeks (day ⫺7 to day 14). Injections of amyloid-enhancing factor (AEF) and Freund’s complete adjuvant (CFA) were given on day 0 and day 1, respectively. Blood samples were obtained at the indicated intervals from each mouse. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05, ⴱⴱ ⫽ P ⬍ 0.005, and ⴱⴱⴱ ⫽ P ⬍ 0.0005 versus mice that received 0% fenofibrate. (Table 2). Among subgroups of the AEF/CFA⫹ group, the mean IL-6 level was lowest in mice that received 0.2% fenofibrate. However, there was remarkable individual variation in IL-6 levels in the same subgroup. Thus, we failed to show a significant correlation between the dose of fenofibrate and the plasma IL-6 level or between the plasma IL-6 level and the SAA concentration. Fenofibrate decreased plasma triglyceride levels on day 14 in both the AEF/CFA⫺ and AEF/CFA⫹ groups. In the AEF/CFA⫺ group, the triglyceride concentration was 89% lower in mice that received 0.2% fenofibrate than in those that received 0% fenofibrate (Table 3). In the AEF/CFA⫹ group, the triglyceride concentration was 57–70% lower in the fenofibratetreated mice than in those that received 0% fenofibrate. Unlike reductions in amyloid deposits or SAA concenTable 2. Plasma interleukin-6 (IL-6) concentrations on day 14* Group, fenofibrate dose (n) AEF/CFA⫺ 0% (7) 0.2% (7) AEF/CFA⫹ 0% (7) 0.05% (7) 0.1% (7) 0.2% (7) IL-6, pg/dl 17 ⫾ 2 19 ⫾ 2 2,375 ⫾ 4,099 1,764 ⫾ 2,581 3,297 ⫾ 2,615 1,146 ⫾ 1,776 * Values are the mean ⫾ SEM (see Table 1 for other definitions). trations, the reduction in triglyceride concentrations almost reached the plateau in the mice that received 0.05% fenofibrate, the minimum dose in this experiment. DISCUSSION This study demonstrates that fenofibrate inhibits reactive AA amyloidosis by reducing SAA in an inflammatory murine model. Histologic examination clearly showed that fenofibrate suppressed amyloid deposits in the spleen in the AEF/CFA⫹ group (Figure 2). The higher the dose of fenofibrate, the lower the severity score of amyloid deposits in the spleen (Table 1). Corresponding to the severity of the amyloid deposits, fenofibrate dose-dependently suppressed the elevated SAA concentrations induced by inflammatory stimuli. Furthermore, fenofibrate decreased macroscopic inflammatory responses such as massive ascites and intraperitoneal adhesions. Although fenofibrate has been widely used in Europe since 1975, its mechanism of action has only recently been elucidated in detail. In 1990, peroxisome proliferator–activated receptor ␣ (PPAR␣) was found to be a ligand-dependent transcription factor (20). Fibrates are ligands for this receptor, and beneficial effects of fenofibrate on lipid metabolism are exerted via PPAR␣ activation. Subsequent studies have clarified the role of PPAR␣ in regulating lipid metabolism and inflammation control via different pathways. In the main pathway, PPAR␣ binds to its ligand and dimerizes with the retinoic acid receptor (RXR). This PPAR␣–RXR heterodimer binds to DNA motifs (termed PPARresponsive elements) in the promoters of many target genes, particularly those implicated in lipid metabolism. Triglyceride reduction is caused by the up-regulation of lipoprotein lipase and the enzymes involved in EFFECT OF FENOFIBRATE ON AMYLOIDOSIS ␤-oxidation and down-regulation of apolipoprotein C-III (21,22). In another pathway, PPAR␣ exerts antiinflammatory activities by interfering with transcription factors, such as nuclear factor B (NF-B) and activator protein 1 (15,23). NF-B is known to be an important redox-sensitive transcription factor that regulates the transcription of genes encoding inflammatory cytokines (including TNF␣, IL-1, and IL-6), adhesion molecules, and chemokines (24,25). Fenofibrate may reduce inflammatory mediators that stimulate SAA production by negatively regulating NF-B signaling pathways. In this study, a sufficient reduction of triglyceride levels was observed in mice that received even 0.05% fenofibrate (Table 3), while a reduction in the SAA concentration (Figure 3) and inhibition of amyloid deposition (Table 1 and Figure 2) continued at higher doses of fenofibrate. In addition, the reduction in SAA concentration and inhibition of amyloid deposition paralleled the improvement in ascites and peritoneal adhesions. These observations are consistent with our hypothesis. In this study, we failed to show a significant correlation between IL-6 and SAA concentrations. This may be due to the differences in kinetics between IL-6 and SAA in the circulation, especially in the acute inflammatory condition. In fact, other investigators have also found that the plasma SAA concentration was not always correlated with the IL-6 concentration in vivo (26,27). Although fenofibrate has a striking preventive effect on reactive amyloidosis in this mouse model, its clinical efficacy in humans with reactive amyloidosis remains to be determined. Currently, the best way to prevent or delay progressive AA amyloid deposition is to reduce SAA concentrations by decreasing inflammatory activity. In a study of patients with systemic AA amyloidosis, not only was the prevention of further accumulation noted, but regression of the amyloid deposits was also observed in the group with low SAA concentrations (28). Moreover, in this group, even amyloid-related organ dysfunction and long-term survival were improved (28). Fenofibrate has been used safely for more than 25 years in patients with hyperlipidemia (29,30). In humans, PPAR␣ activation by fibrates does not cause the peroxisome proliferation–related changes in the liver that result in hepatomegaly and liver cancer in rodents (31,32). 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